Kallikrein is a group of proteases widely distributed in the plasma and tissues of animals, and is known to participate in an enzyme reaction system called the plasma kallikrein-kinin system. An enzymatic system known as the plasma kallikrein-kinin system acts in vivo. It has a close relationship with various other enzymatic reaction systems such as the renin-angiotensin system, the blood clotting system, the fibrinolysis system, the complement system as well as the catecholamine and arachidonic acid cascades, which are mainly related to prostaglandins, leukotrienes and thromboxanes. Accordingly, the kallikrein-kinin system is closely associated with blood pressure regulating action and blood clotting-fibrinolysis-complement system action. Bioregulation and an improving action for peripheral circulation by various physiologically active substances produced by an arachidonic acid cascade are also related to the plasma kallikrein-kinin system. The plasma kallikrein-kinin system plays an important role in the regulation of functions in vivo.
Kinins, such as bradykinin, are produced in the plasma kallikrein-kinin system. They exhibit various physiological actions such as a decrease in blood pressure due to dilation of peripheral blood vessels, promotion of permeability of blood vessels, contraction or relaxation of smooth muscle, induction of pain, induction of inflammation, migration of leucocytes, liberation of catecholamine from the adrenal cortex, etc. Kinins are also known as mediators in acute inflammations, including allergic reactions, whereby their existence in vivo has a profound significance.
The plasma kallikrein-kinin system involves a series of enzyme reactions. Within the plasma kallikrein-kinin system, it is believed that a blood coagulation factor XII (a Hageman factor, hereinafter abbreviated as FXII) is activated in vivo by injury and invasive stimulation to tissues whereby a series of enzymatic reaction systems results. Thus, the activated blood coagulation factor XII (FXIIa) acts on the plasma prekallikrein (hereinafter abbreviated as PK) which exists in the same plasma to convert it to a plasma kallikrein which is an enzyme in an activated form. Then the plasma kallikrein acts on a high-molecular-weight kininogen (hereinafter abbreviated as HK) to liberate bradykinin, which is a nonapeptide.
Kinins such as bradykinin, which are liberated by the enzymatic reaction of the plasma kallikrein-kinin system, exhibit various physiological actions as mentioned already. Accordingly, substances which inhibit the action of bradykinin or substances which inhibit the production of bradykinin by interfering with the formation of kallikrein maybe useful as anti-inflammatory, analgesic and antiallergic agents.
Therefore, establishment of a method for measuring the degree to which substances, compounds or components inhibit or promote the production of kallikrein in a reliable, simple, easy, quick and precise manner is a very important means for ascertaining the action which helps the above-mentioned bioregulation systems. It is also useful for developing drugs for regulating or controlling the bioregulation systems.
When screening or evaluating drugs using plasma prekallikrein-activation are carried out in vitro, activation of prekallikrein through activation of FXII by an invasive stimulation to tissues and injury such as an intravital reaction cannot be conducted. A substance which activates the prekallikrein may be added to an isolated plasma to carry out a reaction which induces plasma kallikrein production in vitro.
However, the plasma of animals contain various components in addition to the above-mentioned components. For example, components which have an effect (such as an inhibition or a promotion) on plasma kallikrein production and other unknown factors are contained in animal plasma. Accordingly, when the activity of the tested substance towards the production of kallikrein is measured utilizing the above-mentioned reaction system with an objective of screening or the like of drugs using the plasma prekallikrein activation system, use of animal plasma per se is complicated by various factors containing unknown components which may affect the plasma kallikrein-kinin reaction system. Consequently, controlling the reaction system is highly technical and complex when animal plasma is used as a source of reactants.
Specific examples of the method for measuring the activity of drugs for suppressing pain, inflammation, allergy, etc. induced by bradykinin, such as an analgesic agent, an anti-inflammatory agent, an antiallergic agent, etc. the above-mentioned drugs are: (1) a method of measuring the inhibiting activity of a test substance to monitor the production of kallikrein using animal plasma, U.S. Pat. No. 4,985,354; (2) a method of measuring the inhibiting and promoting activities of a test substance to the production of FXIIa using animal plasma, U.S. Pat. No. 5,599,683; and (3) a method of measuring the inhibiting and promoting activity of a test substance to the production of FXIIa, kallikrein or bradykinin by a reconstituted plasma kallikrein-kinin system, U.S. Pat. No. 5,648,228. However, as discussed above, test materials containing plasma are impure and interfere with or inhibit kallikrein production.
In U.S. Pat. No. 5,648,228, the reconstituted plasma kallikrein-kinin reaction system for measuring the physiological action of a tested substance combines FXII, PK, and preferably HK, each being substantially purified. However, to produce kallikrein in this system, a heterologous surface having a negative charge (kaolin) is further required to activate FXII and, thus, to activate PK. It would be desirable to produce a prekallikrein-activating substance, produced in living organisms, in a pure or isolated form, in which interfering or competing components (e.g. protease inhibitors) or other factors are substantially or completely absent, for a more faithful reproduction of an actual prekallikrein-activating reaction in living organisms.
Until now, it has been believed that prekallikrein is activated to kallikrein by FXIIa. Thus, all of the above-mentioned methods are based on the prekallikrein-activating system by activation with FXIIa. On the contrary, the present inventors have found a novel prekallikrein-activating system in which FXIIa does not participate. Use of this prekallikrein-activating system makes it possible to carry out the screening of an inhibitor on the production of kallikrein based on a new biological mechanism. In addition, since we can obtain new knowledge or information about the kallikrein-producing system, the prekallikrein-activating method of the present invention is very useful.